The overall objective of the research project is to delineate the sequence of events which occurs when superoxide dismutase (erythrocuprein), which contains two atoms of copper and two atoms of Zn, acts on two molecules of superoxide (O2minus) and two protons to yield a molecule of oxygen and a molecule of hydrogen peroxide. The heart of the project rests with the stability and solubility of the bovine enzyme in mixed organic solvents at temperatures well below O degrees C. It is proposed that under these conditions the overall reaction will be slowed to a point where intermediates in the enzymic reaction can be detected by EPR and optical methods. To this end a low-temperature rapid-freeze device utilizing a rotating cryostat and a stopped-flow spectrophotometer adapted for low-temperature work will be constructed. These instruments will be of general use in electron-transfer and other enzyme reactions where EPR and optical properties are expressed. In addition, a number of metal ion binding and spectroscopic studies have been proposed which are designed to clarify the interrelationships of the various metal ions in the enzyme molecule.